Bottom Line:
Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner.Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs.Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

Affiliation: Institute of Immunology, Tsinghua University School of Medicine, Beijing 100084, China.

ABSTRACTCD1d-dependent NKT cells have been extensively studied; however, the function of CD8(+)NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8(+)NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8(+)NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8(+)NKT-like cell development is normal in CD1d(-/-) mice, which suggests that CD8(+)NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8(+)NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

f6: CD8+NKT-like cells involved in immune downregulation in vivo.(a) NKTGFP or NKTOVA cells were injected into naïve mice, which subsequently received immunization with GFP-DCs and OVA-DCs. Two weeks later, splenocytes from each group were co-cultured with GFP-DCs or OVA-DCs, and the TNF-alpha and IFN-gamma production levels were detected in the supernatant at 72 hours. These data are representative of three independent experiments (n = 8). (b,c) NKTGFP and NKTSV cells were pre-injected into RIP-OVA mice, which was subsequently injected peritoneally with OVA-specific CD8 T cells and GFP-DCs loaded with OT-I peptides. The blood glucose dynamics (b) and body weight (c) were measured in both conditions. These data are representative of two independent experiments (n = 8). ***P < 0.001.

Mentions:
Originally, we showed that GFP-DC immunization increased the number of CD8+ NKTGFP cells in mice. The in vitro data also suggested that CD8+ NKT-like cells might play a role in regulating DCs. Therefore, we further explored the regulatory function of CD8+ NKT-like cells in the immune response in vivo. The mice were first injected with NKTGFP or NKTOVA cells and subsequently received an immunization with both GFP-DCs and OVA-DCs. Two weeks later, splenocytes from each group were co-cultured with GFP-DCs or OVA-DCs for 72 h, and the level of cytokines secreted in the supernatant was determined. We found that, if the mice were pre-treated with NKTGFP, the splenocyte re-challenge by GFP-DCs yielded lower levels of TNF-α and IFN-γ compared with the cells re-challenged by OVA-DCs. Similar results were observed in mice immunized with NKTOVA cells (Fig. 6a). These data demonstrate that intervention with antigen-specific CD8+NKT-like cells kills specific antigen-bearing DCs and reduces their opportunity to stimulate immunocytes and down-regulate the immune response. RIP-OVA transgenic mice were also used to study the immunosuppressive effect of CD8+NKT-like cells. RIP-OVA transgenic mice, which express high levels of ovalbumin protein in their pancreas, were subjected to severe pancreatic islet damage when injected with CTLs activated by OVA257–264-loaded GFP-DCs and exhibited elevated blood glucose levels as well as lower body weight. However, pre-injection with NKTGFP, but not NKTSV, significantly inhibited the blood glucose increase (Fig. 6b) and body weight decrease (Fig. 6c).

f6: CD8+NKT-like cells involved in immune downregulation in vivo.(a) NKTGFP or NKTOVA cells were injected into naïve mice, which subsequently received immunization with GFP-DCs and OVA-DCs. Two weeks later, splenocytes from each group were co-cultured with GFP-DCs or OVA-DCs, and the TNF-alpha and IFN-gamma production levels were detected in the supernatant at 72 hours. These data are representative of three independent experiments (n = 8). (b,c) NKTGFP and NKTSV cells were pre-injected into RIP-OVA mice, which was subsequently injected peritoneally with OVA-specific CD8 T cells and GFP-DCs loaded with OT-I peptides. The blood glucose dynamics (b) and body weight (c) were measured in both conditions. These data are representative of two independent experiments (n = 8). ***P < 0.001.

Mentions:
Originally, we showed that GFP-DC immunization increased the number of CD8+ NKTGFP cells in mice. The in vitro data also suggested that CD8+ NKT-like cells might play a role in regulating DCs. Therefore, we further explored the regulatory function of CD8+ NKT-like cells in the immune response in vivo. The mice were first injected with NKTGFP or NKTOVA cells and subsequently received an immunization with both GFP-DCs and OVA-DCs. Two weeks later, splenocytes from each group were co-cultured with GFP-DCs or OVA-DCs for 72 h, and the level of cytokines secreted in the supernatant was determined. We found that, if the mice were pre-treated with NKTGFP, the splenocyte re-challenge by GFP-DCs yielded lower levels of TNF-α and IFN-γ compared with the cells re-challenged by OVA-DCs. Similar results were observed in mice immunized with NKTOVA cells (Fig. 6a). These data demonstrate that intervention with antigen-specific CD8+NKT-like cells kills specific antigen-bearing DCs and reduces their opportunity to stimulate immunocytes and down-regulate the immune response. RIP-OVA transgenic mice were also used to study the immunosuppressive effect of CD8+NKT-like cells. RIP-OVA transgenic mice, which express high levels of ovalbumin protein in their pancreas, were subjected to severe pancreatic islet damage when injected with CTLs activated by OVA257–264-loaded GFP-DCs and exhibited elevated blood glucose levels as well as lower body weight. However, pre-injection with NKTGFP, but not NKTSV, significantly inhibited the blood glucose increase (Fig. 6b) and body weight decrease (Fig. 6c).

Bottom Line:
Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner.Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs.Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.

Affiliation:
Institute of Immunology, Tsinghua University School of Medicine, Beijing 100084, China.

ABSTRACTCD1d-dependent NKT cells have been extensively studied; however, the function of CD8(+)NKT-like cells, which are CD1d-independent T cells with NK markers, remains unknown. Here, we report that CD1d-independent CD8(+)NKT-like cells, which express both T cell markers (TCRβ and CD3) and NK cell receptors (NK1.1, CD49b and NKG2D), are activated and significantly expanded in mice immunized with GFP-expressing dendritic cells. Distinct from CD1d-dependent NKT cells, CD8(+)NKT-like cells possess a diverse repertoire of TCRs and secrete high levels of IFN-gamma but not IL-4. CD8(+)NKT-like cell development is normal in CD1d(-/-) mice, which suggests that CD8(+)NKT-like cells undergo a unique development pathway that differs from iNKT cells. Further functional analyses show that CD8(+)NKT-like cells suppress T-cell responses through elimination of dendritic cells in an antigen-specific manner. Adoptive transfer of antigen-specific CD8(+)NKT-like cells into RIP-OVA mice prevented subsequent development of diabetes in the animals induced by activated OT-I CD8 T cells. Our study suggests that CD8(+)NKT-like cells can function as antigen-specific suppressive cells to regulate the immune response through killing antigen-bearing DCs. Antigen-specific down regulation may provide an active and precise method for constraining an excessive immune response and avoiding bypass suppression of necessary immune responses to other antigens.